The effect of pH, bile and calcium on the adhesion ability of probiotic enterococci of animal origin to the porcine jejunal epithelial cell line IPEC-J2.

Examination of adhesion ability using a quantitative assay based on radiolabelled bacteria showed that 10 Enterococcus strains exhibited adhesion ability from 2 to 4%. Enterococcus faecium EF2019 (isolate from rabbit faeces, deponed to Czech Culture of Microorganisms in Brno, CCM 7420) showed the highest adhesion ability (4.0+/-0.4%). With regard to survival, all strains displayed good resistance towards 0.3% oxgall and HCl (pH 3.0). Pretreatment of strains with HCl (pH 3.0) significantly reduced their adhesion. Pretreatment of strains by oxgall significantly reduced the adhesion capacity of E. faecium EF2019, EF1839 and EF319 strains, while the adhesion ability of E. faecium EE3 (isolate from canine feed) slightly increased. Furthermore, addition of calcium (200 mmol/l) significantly increased (P<0.001) the adhesion ability for all strains tested. The adhesion ability of the isolates from rabbits, EF1839 and EF529, as well as the isolate EE3 (strain from canine feed) increased from 2-3% up to 50-55% upon calcium addition. Despite, in general low adhesive properties, strains can survive passage through the gastrointestinal tract.

Cereulide formation by Bacillus weihenstephanensis and mesophilic emetic Bacillus cereus at temperature abuse depends on pre-incubation conditions.

Emetic toxin (cereulide) formation was recently identified in a psychrotolerant species, Bacillus weihenstephanensis [Thorsen, L., Hansen, B.M., Nielsen, K.F., Hendriksen, N.B., Phipps, R.K., Budde, B.B., 2006. Characterization of emetic Bacillus weihenstephanensisis, a new cereulide-producing bacterium. Applied and Environmental Microbiology, 72, 5118-5121.]. Although recent findings indicated B. weihenstephanensis as a cereulide producer only limited information is available regarding environmental conditions affecting cereulide production. In the present study a model agar system was used to compare cereulide production during surface growth of B. weihenstephanensis MC67, and two well known mesophilic cereulide producing Bacillus cereus strains, NC7401 and NS117. Cereulide production was quantified by use of Liquid-Chromatography Mass Spectrometry/Mass Spectrometry. Cereulide production of B. weihenstephanensis MC67 occurred in stationary growth phase, as previously observed for B. cereus, and biomass formation and cereulide formation showed a linear correlation. During incubation at 5 degrees C for 1, 2 and 3 weeks growth was inhibited and as a consequence no detectable cereulide production occurred for any of the three strains. Similar results were obtained for the mesophilic B. cereus strains when incubated at 8 degrees C, whereas B. weihenstephanensis MC67 grew to stationary phase and produced 0.002 microg cereulide/cm(2) agar surface in 1 week. Raising the temperature from 5 degrees C to 25 degrees C for 24 h after 1 week of incubation resulted in growth to stationary phase and production of variable levels of cereulide. B. weihenstephanensis MC67 produced 6.18 microg cereulide/cm(2), B. cereus NS117 0.91 microg cereulide/cm(2) and B. cereus NC7401 0.09 microg cereulide/cm(2). Similar levels of cereulide was produced by the mesophilic strains when raising the temperature from 8 degrees C (instead of from 5 degrees C) to 25 degrees C for 24 h, while a considerably lower level was produced by B. weihenstephanensis MC67 (0.10 microg cereulide /cm(2)). If the temperature was raised from 5 degrees C and 8 degrees C to 25 degrees C for 24 h after an increased incubation time for 2 and 3 weeks, all three strains produced considerably less cereulide. B. weihenstephanensis MC67 produced 100-6000 times less and the mesophilic B. cereus strains produced 9-40 times less cereulide. These results can partly be explained by differences in the growth at the temperature abuse. Effect of chill storage on cereulide production at temperature abuse has not been investigated previously. Results of the present study indicate that storage at 5 and 8 degrees C will not lead to emetic intoxications, however the time at, and choice of chill temperature will determine the amount of cereulide produced in a temperature abuse situation. These results are of relevance for the safety of chilled foods of extended durability.

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage.

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 degrees C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO2. The sensitivity to 20% CO2 was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO2 was combined with 2% O2 and in 3 weeks when combined with "0"% O2 (the remaining atmosphere was made up from N2). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m(2)/24 h and the atmospheres were 2% O2/20% CO2 and "0"% O2/20% CO2. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h and "0"% O2/20 % CO2 inhibited growth of the three strains during 4 weeks storage at 8 degrees C. Cereulide production was undetectable during storage at 8 degrees C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 degrees C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 degrees C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 degrees C for 1 week in MAP with OTR of 1.3 or 40 ml/m(2)/24 h and 2% O2 resulted in cereulide concentrations of 0.816-1.353 microg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h, "0"% O2/20 % CO2 resulted in minimal cereulide production (0.004 microg/g meat sausage) at abuse condition. Extension of MAP storage at 8 degrees C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production. Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 degrees C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.

The effect of reuterin on the lag time of single cells of Listeria innocua grown on a solid agar surface at different pH and NaCl concentrations.

The lag time of single cells of Listeria innocua grown on the surface of Brain Heart Infusion Agar was studied by microscopy and image analysis. An experimental set-up that enabled relocation of the cells on the agar surface was developed and used to collect data from 50 to 100 individual cells at a time. Reuterin was added at different concentrations (0-10 AU/ml) and it was observed that it increased both the lag time of the cells and its variance. Furthermore, for a large proportion of cells, reuterin completely prevented the cell division within the time of observation. Reuterin in combination with low pH inhibited the cell division even more efficiently. A similar effect was observed for the combination of reuterin and sodium chloride. Our experimental set-up provides a good model system for generating data on the lag time of single cells on solid surfaces, which can improve the predictions of microbial growth on solid food matrices.

Characterization of emetic Bacillus weihenstephanensis, a new cereulide-producing bacterium.

Cereulide production has until now been restricted to the species Bacillus cereus. Here we report on two psychrotolerant Bacillus weihenstephanensis strains, MC67 and MC118, that produce cereulide. The strains are atypical with regard to pheno- and genotypic characteristics normally used for identification of emetic B. cereus strains. MC67 and MC118 produced cereulide at temperatures of as low as 8 degrees C.

Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus employ different strategies to cope with acid stress.

Responses of Listeria innocua and Lactobacillus delbrueckii subsp. bulgaricus to a rapid change in extracellular pH (pHex) from pHex 6 to a range of concentrations down to pHex 3.0 were examined, using HCl and lactic acid (LA) as acidulants. A new fluorescent probe 5-(and-6)-carboxy-2', 7'-dichlorofluorescein diacetate succinimidyl ester (CDCFDA-SE) was employed that enabled reliable measurements of intracellular pH (pHi) to a minimum pHi of 4.0. Changes in pHi and H+ fluxes from immobilised bacteria were measured using fluorescence ratio imaging microscopy (FRIM) and a non-invasive ion flux measuring technique (MIFE), respectively. L. innocua maintained a relatively constant pHi of 5.5-6.1 at pHex 4 and 5 via H+ extrusion. In contrast, L. delbrueckii subsp. bulgaricus progressively lowered pHi towards pHex over the entire pHex range examined. The type of acidulant used influenced pH regulation with both pHi and H+ -fluxes being more severely affected by LA compared to HCl. Overall, our data demonstrated different adaptive strategies in these two bacteria. While L. innocua expels protons to maintain a constant pHi, L. delbrueckii subsp. bulgaricus allows proton entry after acidic treatment so that pHi follows pHex.

Leucocins 4010 from Leuconostoc carnosum cause a matrix related decrease in intracellular pH of Listeria monocytogenes.

A mixed culture of single cells of Listeria monocytogenes and the bacteriocin producing Leuconostoc carnosum 4010 showed growth inhibition of L. monocytogenes, although the intracellular pH (pHi) of L. monocytogenes followed by fluorescence ratio imaging microscopy was not affected. Furthermore, L. monocytogenes was exposed to the bacteriocins leucocins 4010 and nisin either in a liquid filled chamber or on the surface of an agar containing bacteriocins. Both bacteriocins caused dissipation of the pH gradient in L. monocytogenes and the effect was clearly dependent on the matrix, as the decrease in pHi occurred much more rapidly in liquid than in agar.

The survival and persistence in the human gastrointestinal tract of five potential probiotic lactobacilli consumed as freeze-dried cultures or as probiotic sausage.

Among five lactobacilli (L. plantarum MF1291, MF1298, DC13, L. pentosus MF1300 and L. salivarius DC5) which were administrated as freeze-dried cultures for 17 volunteers, MF1298 and DC13 were the most frequently reisolated strains in faeces demonstrating the human gastric survival of these strains. Furthermore, MF1298 and DC13 persisted in the same volunteer after ended intake, suggesting host-specific persistence behaviour. When MF1298 was administrated as sausage fermented with this strain, the number of volunteers harbouring MF1298 increased from 4 to 10 indicating that the sausage matrix protects the survival through the gastrointestinal tract (GIT).

Two subpopulations of Listeria monocytogenes occur at subinhibitory concentrations of leucocin 4010 and nisin.

In situ analyses of single Listeria monocytogenes cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (DeltapH), and the other consisted of cells that maintained DeltapH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate DeltapH for 90% of the cell population (ED90) was predicted. ED90 increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED90 decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED90. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.

Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity.

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.

Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages.

Potential probiotic cultures suitable as starter cultures for the Scandinavian-type fermented sausages were identified among strains well-adapted to fermented meats as well as strains originating from a culture collection. From 15 different fermented meat products, 22 strains were isolated as dominant non-starter lactic acid bacteria (NSLAB). The isolates were identified by RAPD, API and sequence analysis of 16S rRNA and showed to be five strains of Lactobacillus sakei, five strains of Lactobacillus farciminis, five strains belonging to the group of Lactobacillus plantarum/pentosus, four strains of Lactobacillus alimentarius, two strains of Lactobacillus brevis and one strain of Lactobacillus versmoldensis. Heterofermentative strains as well as strains not growing at 37 degrees C and not lowering pH below 5.1 in a meat model were excluded leaving 9 strains for further studies. These strains together with 19 strains from a culture collection were evaluated by in vitro methods including survival upon exposure to pH 2.5 or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. Strains that fulfilled all the probiotic criteria and showed to be fast acid producers in a meat model included three strains belonging to the group of Lb. plantarum/pentosus (MF1291, MF1298, MF1300) which originated from the dominant NSLAB of fermented meat products. MF1291 and MF 1298 were further identified as Lb. plantarum and MF1300 as Lb. pentosus. The three strains were all successfully applied as starter cultures for the production of fermented sausage. The viable count at the end of the processing period reached high cell numbers (4.7x10(7)-2.9x10(8) cfu/g) and pH of the sausages decreased to pH 4.8-4.9 without any flavour deviation compared to sausage fermented by a commercial meat starter culture.

Osmotic stress leads to decreased intracellular pH of Listeria monocytogenes as determined by fluorescence ratio-imaging microscopy.

Intracellular pH (pH(i)) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH(i), and recovery on solid medium was impaired compared to that in liquid medium. N,N'-dicyclohexylcarbodiimide abolished pH(i) recovery, and lowering a(w) with glycerol showed no effect on pH(i).

The antilisterial effect of Leuconostoc carnosum 4010 and leucocins 4010 in the presence of sodium chloride and sodium nitrite examined in a structured gelatin system.

To further enhance biopreservation of meat products, the antilisterial effect of the newly described protective culture Leuconostoc carnosum 4010 and its bacteriocins, leucocins 4010, was examined in the presence of sodium chloride and sodium nitrite in a solid matrix using a structured gelatin system. Interaction between Listeria monocytogenes 4140 and Leuc. carnosum 4010 or the leucocins 4010-resistant mutant L. monocytogenes 4140P showed that the inhibitory effect of Leuc. carnosum 4010 in the gelatin system was caused by the production and activity of leucocins 4010. The presence of sodium chloride (2.5% w/v) and sodium nitrite (60 mg/l) reduced the antilisterial effect of Leuc. carnosum 4010 in the structured gel system compared to the use of Leuc. carnosum 4010 alone. Investigations carried out at 10 degrees C showed that the lag phase of L. monocytogenes 4140 in the presence of Leuc. carnosum 4010 was reduced from 71 to 58 h by the addition of sodium chloride and to 40 h by the addition of sodium nitrite. Addition of sodium chloride increased the maximum specific growth rate of L. monocytogenes 4140 in the presence of Leuc. carnosum 4010 from 0.02 to 0.06 h(-1), whereas no change was observed by the addition of sodium nitrite. Compared to the antilisterial effect of leucocins 4010 alone, the addition of sodium chloride (2.5%, w/v) decreased the antilisterial effect at high concentrations of leucocins 4010 (5.3 and 10.6 AU/ml) as measured after 11 days of incubation at 10 degrees C. In gels with added leucocins 4010, the most pronounced reduction in growth of L. monocytogenes 4140 was observed at the highest concentration of leucocins 4010 (10.6 AU/ml) together with sodium nitrite (60 mg/l). More detailed information on the lag phase and the maximum specific growth rate of single colonies of L. monocytogenes 4140 in the presence of leucocins 4010 was obtained using microscopy and image analysis. No pronounced difference in the growth of single colonies was observed in the gel system. Real-time measurements of colony growth at 10 degrees C in the gelatin matrix showed that the growth inhibiting effect of leucocins 4010, including a longer lag phase as well as a lower maximum specific growth rate for L. monocytogenes 4010, was negated in the presence of 2.5% (w/v) sodium chloride.

Leuconostoc carnosum 4010 has the potential for use as a protective culture for vacuum-packed meats: culture isolation, bacteriocin identification, and meat application experiments.

A new culture, Leuconostoc carnosum 4010, for biopreservation of vacuum-packed meats is described. The culture originated from bacteriocin-producing lactic acid bacteria (LAB) naturally present in vacuum-packed meat products. Approximately, 72,000 colonies were isolated from 48 different vacuum-packed meat products and examined for antibacterial activity. Bacteriocin-producing colonies were isolated from 46% of the packages examined. Leuc. carnosum was the predominant bacteriocin-producing strain and Leuc. carnosum 4010 was selected for further experiments because it showed strong antilisterial activity without producing any undesirable flavour components in meat products. For identification of the bacteriocins produced, partial purification was carried out by ammonium sulphate precipitation, dialysis, and cation exchange chromatography. SDS-PAGE analysis revealed two bands with inhibitory activity corresponding to molecular sizes of 4.6 and 5.3 kDa. N-terminal amino acid sequencing showed that Leuc. carnosum 4010 produced two bacteriocins highly similar or identical to leucocin A and leucocin C. Application experiments showed that the addition of 10(7) cfu/g Leuc. carnosum 4010 to a vacuum-packaged meat sausage immediately reduced the number of viable Listeria monocytogenes cells to a level below the detection limit and no increase of L. monocytogenes was observed during storage at 5 degrees C for 21 days. The results presented demonstrate that Leuc. carnosum 4010 is suitable as a new protective culture for cold-stored, cooked, sliced, and vacuum-packed meat products.

Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy.

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.

Responses of Listeria monocytogenes to acid stress and glucose availability monitored by measurements of intracellular pH and viable counts.

Physiological aspects of the response of Listeria monocytogenes to acidic conditions and effect of glucose availability were studied by fluorescence ratio-imaging microscopy (FRIM) as compared with traditional viable counts. Three types of experiments were conducted: (i) static with measurements of intracellular pH (pHi) at extracellular pH (pHo) values ranging from pH 3.0 to 6.0 at 0.5 pH unit intervals; (ii) kinetic with monitoring of bacterial responses to changes in the pHo from the value of 6.0 to 4.0 or 3.0; (iii) survival experiments studying bacterial recovery in response to a shift to favourable conditions after a treatment at low pH. All the experiments were performed at three levels of glucose in the medium (0, 1, and 10 mM). Both survival and pHi were greatly affected by pHo and glucose availability with the highest values for CFU and pHi at highest glucose concentration and pHo values in the medium in all trials. A high correlation (R2 = 0.995) between pHi and CFU counts was observed. The pH gradient started to collapse at pHo 4 and below for trials with glucose in the medium and at pHo 5.5 and below without glucose. A recovery step was proposed after the apparently lethal treatment to assess cell viability by FRIM.

Responses of Listeria monocytogenes to acid stress and glucose availability revealed by a novel combination of fluorescence microscopy and microelectrode ion-selective techniques.

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H(+) at either side of the bacterial membrane in real time and therefore to evaluate the rate of H(+) flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH(o)) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH(i)) and net H(+) fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH(i) remained above 7.0 within a pH(o) range of 4 to 6 and decreased below pH(o) 4. Above pH(o) 4, H(+) extrusion increased correspondingly, with the maximum value at pH(o) 5.5, and below pH(o) 4, a net H(+) influx was observed. Without glucose in the medium, the pH(i) decreased, and a net H(+) influx was observed below pH(o) 5.5. A high correlation (R = 0.75 to 0.92) between the pH(i) and net H(+) flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH(i) homeostasis in L. monocytogenes subjected to acid stress.